The mechanism by which chemicals such as carcinogens induce frameshift mutations is not well understood. This lack of knowledge is especially evident in the determination of what structural features of the chemicals are required for high mutagenic activity and why certain sites on the DNA are especially sensitive to frameshift mutagens. Several events are required for such a mutation to occur: (1) uptake of the chemical into the cell; (2) activation, if not already reactive, to an electrophilic species by enzymes; (3) transport to and association with the DNA; (4) formation of a covalent bond or complex with the DNA; (5) recognition of the lesion by repair or replication enzymes; (6) misrepair or misreplication of the lesion, resulting in the induction of a frameshift mutation. We plan to investigate the last three steps of this process using newly developed methods of in vitro biochemistry. These new methods are: (1) direct DNA sequencing for determination of nucleoside and sequence specificity of the reaction forming a mutagen-DNA covalent bond; (2) cloning of DNA fragments containing specific DNA lesions produced in vitro; and (3) treatment of DNA fragments containing covalently bound mutagens with isolated repair endonucleases. Using these tools, we will be able to study the molecular events leading to a frameshift mutation at a much higher resolution than was previously possible.